Analysis of Break-Induced Replication

描述（由申请人提供）： 断裂染色体的修复对于维持染色体结构和基因组稳定性至关重要。本研究的重点是阐明断裂诱导复制（BIR）修复双链染色体断裂（DSB）的机制。BIR在重新启动停滞和断裂的DNA复制叉、在没有端粒酶的情况下维持端粒以及在新发现的与人类癌症和发育疾病相关的单染色体破碎现象（称为染色体碎裂）中起关键作用。微同源介导的BIR（MM-BIR）过程已被假设为长距离模板转换事件，导致加入遥远的序列，创造新的基因融合。该实验室以前的工作已经确定了复制蛋白Pol 32和PCNA的作用，它们对BIR至关重要，但对模式生物芽殖酵母中的正常复制或其他类型的DSB修复不起作用。继续开展这项研究的拟议项目将侧重于两个主要专题。首先，将追求BIR的分子机制，通过诱导型内切核酸酶产生位点特异性DSB。已经开发了一种新的测定法来研究BIR期间在遥远位置的同源序列之间或同源序列之间（允许研究MM-BIR的高度错配序列）的令人惊讶的频繁模板跳跃。该测定允许人们区分断裂的DNA末端通过链侵入定位并与远端序列重组的方式，以及随后在BIR中复制的链如何跳到第三个位置。将研究错配修复蛋白在阻止分叉序列之间的BIR中的作用。模板跳跃使用与初始链侵入步骤不同的机制的证据将被追求。将探索跳跃不需要Rad 51重组酶蛋白的可能性。将特别关注Rdh 54的作用，Rdh 54是第一个专门为模板跳跃所需的蛋白质，但却能胜任 简单BIR和其它重组事件。通过用CRISPR核酸内切酶切割特定链而产生的断裂复制叉的修复是否遵循与迄今为止研究的异位模型系统和端粒修复事件相同的规则是一个根本性的重要问题。第二个主要目标将是检查易位和重排，其中在修复连接处几乎没有同源性。将使用一种新的检测方法，该方法涉及通过连接远距离序列来创建功能性内含子。最后，将通过DNA测序回收并分析模板跳跃到不相关序列中，以便更好地定义微同源性介导的事件需要多少同源性和相邻同源性。这些研究对于理解与人类疾病相关的染色体重排的起源具有非常重要的意义，包括由非相互易位形成引起的杂合性丢失，节段复制和经历chromothripsis的染色体中的惊人重排。

DESCRIPTION (provided by applicant): Summary The repair of broken chromosomes is essential for maintenance of chromosome structure and genome stability. This proposal focuses on delineating the mechanisms of repair of double-strand chromosome breaks (DSBs) by break-induced replication (BIR). BIR plays a key role in restarting stalled and broken DNA replication forks, in maintaining telomeres in the absence of the telomerase enzyme and in the newly discovered phenomenon of single chromosome shattering, known as chromothripsis, which is associated with human cancers and developmental diseases. A microhomology-mediated BIR (MM-BIR) process has been hypothesized to account for long-distance template switching events that lead to the joining of distant sequences to create novel gene fusions. Previous work from this laboratory has identified roles for replication proteins Pol32 and PCNA that are essential for BIR but not for normal replication or other types of DSB repair in the model organism, budding yeast. The proposed project to continue this study will focus on two main topics. First, the molecular mechanism of BIR will be pursued, creating site-specific DSBs by an inducible endonuclease. A novel assay has been developed to study surprisingly frequent template jumps during BIR both between homologous sequences in distant locations or between homeologous sequences (highly mismatched sequences that permit study of MM-BIR). This assay permits one to distinguish between the way in which a broken DNA end locates and recombines with a distant sequence by strand invasion and how the subsequent replicating strands in BIR then can jump to a third location. The role of mismatch repair proteins in discouraging BIR between diverged sequences will be investigated. Evidence that template jumping uses a different mechanism from the initial strand invasion step will be pursued. The possibility that the jump does not require the Rad51 recombinase protein will be explored. Special attention will be devoted to the role of Rdh54, the first protein that is specifically required for template jumps but competent for simple BIR and other recombination events. Whether repair of a broken replication fork, created by nicking a specific strand with a CRISPR endonuclease, obeys the same rules as the ectopic model systems and telomere-repair events that have so far been studied is a fundamentally important question. A second major goal will be to examine translocations and rearrangements in which there is little or no homology at the repair junctions. A novel assay involving creation o a functional intron by joining distant sequences will be used. Finally, template jumps into unrelated sequences will be recovered and analyzed by DNA sequencing in order to better define how much homology and adjacent homeology is required for microhomology-mediated events. These studies are highly significant in understanding the origins of chromosome rearrangements associated with human disease, including the loss of heterozygosity caused by the formation of nonreciprocal translocations, segmental duplications and the astonishing rearrangements in chromosomes experiencing chromothripsis.